JPH081433B2 - Hapten immunoassay - Google Patents

Description

TECHNICAL FIELD The present invention relates to a hapten immunoassay method. More specifically, it relates to an immunoassay method for hapten in serum or plasma.

[Prior Art] Conventionally, as a hapten immunoassay, a method in which a hapten in a sample and a labeled hapten are simultaneously competitively reacted with an antibody has been known (for example, Roy F Etol Clin. Chem. 24, 1801 (1978)).

[Problems to be Solved by the Invention] However, in the conventional measurement method, since the hapten in the sample and the labeled hapten react with the antibody in the state of contact, when measuring a special sample, the serum component in the sample is In some cases, the enzyme activity or the expression of luminescence or fluorescence is significantly inhibited by the influence of. As a result, abnormal measurement values occur, which causes a serious problem that the measurement results do not match the clinical findings.

[Means for Solving the Problems] The present inventors have arrived at the present invention as a result of earnestly examining an immunoassay method for a hapten that does not have the problem of abnormal measurement values. That is, the present invention provides an immunoassay method for quantifying hapten in serum or plasma, comprising: (1) Serum or plasma together with a buffer solution containing a dissociating agent that dissociates the bond between the hapten contained in serum or plasma and the carrier protein. Contacting with an antibody to the insolubilized hapten to react and form a complex (D) of the antibody to the insolubilized hapten and the hapten, (2) washing and removing the unreacted material in the step (1) (3) The complex (D) is reacted with and brought into contact with the label of the hapten or its analog, (4) the unreacted label in the step (3) is washed,
(5) The amount of the labeled substance bound to the insolubilized antibody to the hapten is measured, and (6) the amount of the labeled substance measured in the step (5) is correlated with the amount of hapten in serum or plasma. This is a hapten immunoassay method for measuring the amount of hapten.

In the present invention, as the hapten to be measured,
Thyroid hormones (T3, T4), steroid hormones (such as cortisol, estradiol, testosterone, aldosterone, progesterone and their derivatives) and drugs (such as digoxin, digitoxin, diphenylhydantoin, theophylline, morphine).

Carrier proteins that are bound to haptens include thyroid hormone and thyroxine binding protein (TBP), aldosterone, progesterone, and testosterone and sex hormone binding globulin, diphenylhydantoin and albumin, and cortisol and cortisol binding protein.

Hapten exists partially in the blood, but
Most of them exist in a state of being bound to a carrier protein specific to the hapten. In the present invention, the hapten refers to the sum of those existing in a free state and those existing in a state bound to a carrier protein.

In the buffer solution containing a dissociating agent, the dissociating agent is not particularly limited as long as it dissociates the bond between the hapten and the carrier protein. For example, used in measuring T3 and T4 8-
Examples thereof include anilino-1-naphthalene sulfonate and salicylate.

As the buffer solution, barbital buffer solution, phosphate buffer solution,
Examples thereof include borate buffer solution, glutamate buffer solution, glycine-hydrochloric acid buffer solution, Tris-hydrochloric acid buffer solution, but are not particularly limited thereto. Among the buffers, the preferred one is the barbital buffer used in the measurement of T3 and T4, and the glutamate buffer and the glycine-hydrochloric acid buffer in the measurement of sterod hormone.
Further, a phosphate buffer solution and a Tris-hydrochloric acid buffer solution used in drug measurement.

The concentration of the dissociating agent in the buffer is usually 0.005 to 0.5 w / v%, preferably 0.01 to 0.3 w / v% regardless of the type of the dissociating agent.

The pH of the buffer solution has the highest buffering capacity of each buffer solution, and when measuring steroid hormones and drugs,
It is important to select a pH that enhances the ability to dissociate from the carrier protein. That is, preferably, pH 8 to 9 for barbital buffer, pH 6 to 8 for phosphate buffer, pH 7.5 to 8.5 for borate buffer, pH 2 to 5 for glutamate buffer and glycine-hydrochloric acid buffer. , PH 7.5 to 8.5 in Tris-HCl buffer.

Regarding the antibody to the insolubilized hapten, the antibody can be a polyclonal antibody or a monoclonal antibody. Immunized animals that obtain these antibodies are preferably rabbits, goats, sheep, mice, guinea pigs, pigs, and monkeys.

As the antibody to the insolubilized hapten, the one obtained by binding the antibody to this hapten to a solid-phased carrier can be used.

As the solid-phase carrier, a siliceous inorganic carrier [glass (porous, matte glass, etc.), silica gel, bentonite, etc.], magnetic substance, organic polymer compound or polymer carrier (polyethylene, polystyrene, dextran, paper, etc.) And so on. Of these, preferred are glass, organic polymer compounds or polymer carriers.

Examples of the form include beads, tubes, test tubes and the like.

It is also possible to use any of the solid phased carriers which have been subjected to a treatment for increasing the surface area by sandblasting or the like.

As a method of binding the antibody to the solid phase carrier, a method of chemically binding the antibody [silane coupling agent,
Examples include a method of covalently bonding to a carrier using a cross-linking agent (glutaraldehyde, etc.), a method of bonding by physical adsorption, and a method of applying an antibody to a part of the inner surface of a container (test tube, tube, tray, etc.). To be

In order to form a complex (D) of the insolubilized antibody against the hapten and the hapten by contacting the insolubilized antibody against the hapten (C), the buffer solution containing the dissociation agent (B) and the serum or plasma (A) Can be carried out by a method in which (A), (B) and (C) are reacted and contacted in a suitable container at 5 to 50 ° C. for about 5 minutes to 2 days. A complex of the antibody and the hapten insolubilized by contact or reaction is obtained.

Then, unreacted substances in the above reaction [(A), (B)
Etc.) is washed and removed. The washing is usually carried out with a buffer solution such as deionized water, physiological saline or a phosphate buffer solution, and the washing frequency is usually 1 to 10 times.

Then, the complex (D) is reacted with and brought into contact with the labeled substance (E) of the hapten or its analog. Examples of the above hapten analogs include those in which the amino groups of T3 and T4 are modified with an acyl-type or urethane-type protecting group.

Examples of the labeled substance (E) of the hapten or a hapten analogue include a labeled substance with an enzyme, a fluorescent substance, or a luminescent substance. Known enzymes, fluorescent substances, and luminescent substances can be used. Examples of enzymes include horseradish peroxidase, alkaline phosphatase,
β-galactosidase etc .; europium derivatives etc. as fluorescent substances; N-methylacridinium derivatives etc. as luminescent substances. The labeled product can be produced by a method similar to the conventional method.

Regarding reaction and contact, usually, (D) and (E) are reacted and contacted in a suitable container at 5 to 50 ° C. for about 5 minutes to 2 days.

Then, unreacted substances in the above reaction [(D), (E)
Etc.] is washed and removed. The washing is usually carried out with a buffer solution such as deionized water, physiological saline or a phosphate buffer solution, and the washing frequency is usually 1 to 10 times.

The amount of the labeled substance bound to the insolubilized antibody can be measured by reacting it with an appropriate substrate (o-phenylenediamine / hydrogen peroxide, etc.) and measuring the enzyme activity or absorbance, luminescence intensity, and fluorescence intensity. it can.

The amount of the labeled substance measured is inversely proportional to the hapten concentration in serum and plasma, so serum from a suitable calibration curve prepared in advance,
The amount of hapten in plasma can be read and calculated.

[Examples] Hereinafter, the present invention will be further described with reference to Examples, but the present invention is not limited thereto.

Example 1 Measurement of T3 (triiodothyronine) Preparation of anti-T3 antibody-bound frosted glass beads 100 g of sandblasted frosted glass beads was used.
5% 3-aminopropyltriethoxysilane (hereinafter γ-A
Immersed in an acetone solution (abbreviated as PS) for 10 hours to introduce amino groups on the surface of the glass beads. After washing unreacted γ-APS with deionized water, aminated glass beads were immersed in a 5% glutaraldehyde (hereinafter abbreviated as GA) aqueous solution to introduce aldehyde groups on the surface of the glass beads. After washing unreacted GA with deionized water, the beads were washed with 0.05 mg / ml anti-T3.
The antibody was bound to the surface of the glass beads by immersing in the antibody solution. After the reaction is complete, add beads to 0.1% bovine serum albumin, 0.2M
0.02M phosphate buffer (pH7.2) containing sodium chloride
The glass beads were soaked overnight in to obtain anti-T3 antibody-bound glass beads.

Synthesis of enzyme-labeled T3 Toyobo Co., Ltd. peroxidase (hereinafter abbreviated as POD) 10 mg
Was dissolved in 2 ml of 0.02M phosphate buffer (pH 8.0), and
Add 200 μl of a glutaraldehyde aqueous solution at room temperature for 20
Allowed to react for hours. After the reaction is complete, put the reaction solution into a dialysis tube and dialyz with 0.02M phosphate buffer (pH 8.0) as the external solution to remove unreacted glutaraldehyde and activate POD.
I got

Dimethylformamide (hereinafter abbreviated as DMF) (1 ml) was added to 1 mg of T3 (manufactured by Calbio Chemistry), and stirred and dissolved. The entire amount of the T3 / DMF solution was added to the activated POD solution, and the reaction was performed at room temperature for 12 hours.

After completion of the reaction, the reaction mixture was passed through a Sephadex G-25 column, and fractions having both enzyme activity and immunological activity were collected and designated as enzyme-labeled T3.

Preparation of standard sample A standard sample was prepared by adding an appropriate amount of T3 dissolved in DMF to T3 free serum.

Measurement of T3 Concentration in Serum or Plasma POD-labeled T3 obtained in (a) was added to 0.2% BSA, 0.05% 8-anilino-1-naphthalenesulfonate ammonium-containing 0.12
It was diluted with M barbital buffer (pH 8.6) and adjusted to the concentration required for measurement.

(B) Measurement operation 50 μl of standard solution or sample (serum or plasma) in a test tube
0.2% BSA prepared in (a), 0.05%
500 μl of 0.12 M barbital buffer solution (pH 8.6) containing ammonium 8-anilino-1-naphthalene sulfonate was added and well stirred to obtain a uniform solution. One anti-T3 antibody-bound glass bead was added to this and stirred, and then shaken and stirred at 37 ° C. for 15 minutes. After completion of the reaction, the reaction solution was removed by suction, 2 ml of physiological saline was added, and the solution was removed by suction again. This operation was repeated twice to remove various serum components in the sample.

Then, the beads were transferred to a new test tube, 500 μl of the enzyme-labeled antigen T3 solution of (a) was added, and the mixture was shaken and stirred at 37 ° C. for 15 minutes. After the reaction was completed, the reaction solution was removed by suction and physiological saline 2m
l was added and aspirated again. After repeating this operation twice, the antibody-bound glass beads were transferred to another test tube, and a 0.1 M citrate buffer solution (pH 4.8 containing 3 mg / ml o-phenylenediamine and 0.02% hydrogen peroxide as a substrate). ) Was added and the mixture was shaken and stirred at 37 ° C for 15 minutes.

After the reaction was completed, add 3 ml of 1.5N sulfuric acid, and after the reaction was stopped, 492 nm
The absorbance at was measured. The T3 concentration in the sample (serum or plasma) was read from the calibration curve (Fig.-1) in which the standard point and the absorbance at that point were plotted on a graph paper.

Example 2 Measurement of T4 (thyroxine) Preparation of anti-T4 antibody-bonded ground glass beads Sandblasted ground glass beads 100 g 0.5
% 3-aminopropyltriethoxysilane (hereinafter γ-AP
(S) (abbreviated as S) Immerse in an acetone solution for 10 hours to introduce amino groups on the surface of the glass beads. After washing unreacted γ-APS with deionized water, aminated glass beads were immersed in a 5% glutaraldehyde (hereinafter abbreviated as GA) aqueous solution to introduce aldehyde groups on the surface of the glass beads. After washing unreacted GA with deionized water, the beads were washed with 0.05 mg / ml anti-T4.
The antibody was bound to the surface of the glass beads by immersing in the antibody solution. After the reaction is complete, add beads to 0.1% bovine serum albumin, 0.2M
0.02M phosphate buffer (pH7.2) containing sodium chloride
The glass beads were soaked overnight in to obtain anti-T4 antibody-bound glass beads.

Synthesis of enzyme-labeled T4 Peroxidase manufactured by Toyobo (hereinafter abbreviated as POD) 10 mg
Was dissolved in 2 ml of 0.02M phosphate buffer (pH 8.0), and
Add 200 μl of a glutaraldehyde aqueous solution at room temperature for 20
Allowed to react for hours. After the reaction is complete, put the reaction solution into a dialysis tube and dialyz with 0.02M phosphate buffer (pH 8.0) as the external solution to remove unreacted glutaraldehyde and activate POD.
I got

DMF (1 ml) per 1 mg of T4 (Calbio Chemistry)
Was added, and the mixture was stirred and dissolved. The entire amount of the T4 / DMF solution was added to the activated POD solution, and the reaction was performed at room temperature for 12 hours.

After completion of the reaction, the reaction mixture was passed through a Sephadex G-25 column, and fractions having both enzyme activity and immunological activity were collected and designated as enzyme-labeled T4.

Preparation of standard sample A standard sample was prepared by adding an appropriate amount of T4 dissolved in DMF to T4 free serum.

Determination of T4 Concentration in Serum or Plasma POD-labeled T4 obtained in (a) was added to 0.2% BSA, 0.05% 8-anilino-1-naphthalene ammonium sulfonate 0.12
It was diluted with M barbital buffer (pH 8.6) and adjusted to a concentration required for measurement.

(B) Measurement operation 20μ of standard solution or sample (serum or plasma) in a test tube
0.2% BSA prepared in (a), 0.05%
500 μl of 0.12 M barbital buffer solution (pH 8.6) containing ammonium 8-anilino-1-naphthalene sulfonate was added and well stirred to obtain a uniform solution. One anti-T4 antibody-bound glass bead was added to this and stirred, and then shaken and stirred at 37 ° C. for 15 minutes. After completion of the reaction, the reaction solution was removed by suction, 2 ml of physiological saline was added, and the solution was removed by suction again. This operation was repeated twice to remove various serum components in the sample.

Then, the beads were transferred to a new test tube, 500 μl of the enzyme-labeled antigen T4 solution of (a) was added, and the mixture was shaken and stirred at 37 ° C. for 15 minutes. After the reaction was completed, the reaction solution was removed by suction and physiological saline 2m
l was added and aspirated again. After repeating this operation twice, the antibody-bound glass beads were transferred to another test tube, and 0.1 M citrate buffer solution (pH 4.8, containing 3 mg / ml o-phenylenediamine and 0.02% hydrogen peroxide as a substrate). ) Was added and the mixture was shaken and stirred at 37 ° C for 15 minutes.

After the reaction was completed, 3 ml of 1.5N sulfuric acid was added, and after the reaction was stopped, the absorbance at 492 nm was measured. The T4 concentration in the sample was read from the calibration curve in which the standard point and the absorbance at that point were plotted on a graph paper.

[Effects of the Invention] The immunoassay method of the present invention is a method that enables immunoassay of a hapten that is not affected by unknown serum components. That is,
Even when measuring a special sample, it is a method in which the enzyme activity or the expression of luminescence or fluorescence is not significantly inhibited by the influence of serum components in the sample, and the measurement results are consistent with clinical findings. It is a hapten immunoassay without the problem of the occurrence of

[Brief description of drawings]

FIG. 1 is a graph showing the calibration curve of Example 1, and FIG. 2 is a graph showing the correlation of serum measurement values.

Claims (4)

[Claims] 1. An immunoassay for quantifying hapten in serum or plasma, which comprises: (1) a buffer solution containing a dissociation agent that dissociates serum or plasma from the bond between the hapten contained in serum or plasma and a carrier protein. At the same time, it is contacted with an antibody to the insolubilized hapten to react and form a complex (D) of the antibody to the insolubilized hapten and the hapten, and (2) the unreacted material in the step (1) is washed and removed. Then, (3) the complex (D) is reacted with and brought into contact with the label of the hapten or its analog, (4) the unreacted label in the step (3) is washed,
(5) The amount of the labeled substance bound to the insolubilized antibody to the hapten is measured, and (6) the amount of the labeled substance measured in the step (5) is correlated with the amount of hapten in serum or plasma. A hapten immunoassay for measuring the amount of hapten. 2. The assay method according to claim 1, wherein the hapten is selected from the group consisting of thyroid hormone, steroid hormones and drugs. 3. The assay method according to claim 1, wherein the labeled substance is labeled with an enzyme, a fluorescent substance, or a luminescent substance. 4. The antibody to the insolubilized hapten is an antibody covalently bonded or physically adsorbed to glass beads or polymer beads, or an antibody coated on a part of the inner surface of a container. The measurement method described.